Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Full citations for those references that are numbered can be found at the end of the specification. Each citation is incorporated herein as though set forth in full.
The rapid developments in recombinant DNA techniques have resulted in the identification and isolation of many novel genes, some of known function and some of unknown function. Invariably there is a need to express the gene in a heterologous cell system in order to produce material for structure-function studies, diagnostic reagents such as monoclonal or polyclonal antibodies and material for in vivo activity testing and therapy.
Several alternative systems for the expression of foreign genes have been developed including systems based upon mammalian cells, insect cells, fungal cells, bacterial cells and transgenic animals or plants. The choice of expression system for a given gene depends upon the likely features of the encoded protein, for example any post-translational protein modifications needed for biological activity, as well as the objective of the study. Other important considerations for the investigator are the facilities available, time and cost involved in generating the amounts of recombinant protein required.
The most widely used and convenient system for the production of foreign proteins remains that based on the prokaryote Escherichia coli. The advantages of this system comprise the ease of gene manipulation, the availability of reagents including gene expression vectors, the ease of producing quantities of protein (up to a gram in simple shake-flask culture), speed and the high adaptability of the system to express a wide variety of proteins.
Unfortunately, however, a problem encountered with E. coli based expression systems is the difficulty of producing material which is acceptable for therapeutic use. Some heterologous gene products fail to attain their correct three-dimensional conformation in E. coli while others become sequestered in large insoluble aggregates or “inclusion bodies” when overproduced. Major denaturant-induced solubilization methods followed by removal of the denaturant under conditions that favor refolding are often required to produce a reasonable yield of the recombinant protein. Selection of ORFs from structural genomics projects have also shown that only about 20% of the genes expressed in E. coli render proteins that were soluble or correctly folded. These numbers are startlingly disappointing especially given that most scientists rely on E. coli for initial attempts to express gene products. Furthermore, the use of complex media, antibiotic selection and potentially hazardous inducers may potentially render products such as recombinant antibody fragments produced by E. coli fermentation technology unacceptable to the regulatory authorities for clinical applications. Evidence demonstrating clearance of these agents from the final product must be provided in order to secure regulatory clearance. Clearance of these agents, and especially demonstrating such clearance, is expensive. It is therefore desirable that an expression system should avoid the three above-mentioned problems.
Expression in mammalian cells is often preferred for manufacturing of therapeutic proteins, since post-translational modifications in such expression systems are more likely to resemble those occurring on endogenous proteins in a mammal, than the type of post-translational modifications that occur in microbial expression systems
Several vectors are available for expression in mammalian hosts, each containing various combinations of cis- and in some cases trans-regulatory elements to achieve high levels of recombinant protein in a minimal time frame. However, despite the availability of numerous such vectors, the level of expression of a recombinant protein achieved in mammalian systems is often lower than that obtained with a microbial expression system. Additionally, because only a small percentage of cloned, transfected mammalian cells express high levels of the protein of interest, it can often take a considerably longer time to develop useful stably transfected mammalian cell lines than it takes for microbial systems.
There are a number of reasons for the lack of efficient recombinant protein expression in a host, including, for example, short half life, improper folding or compartmentalization and codon bias. While the Human Genome project has successfully created a DNA “map” of the human genome, the development of protein expression technologies that function uniformly in different expression platforms and for all the protein motifs has not yet been achieved. It would be desirable and advantageous to have alternative stable expression systems available when other systems for protein expression are unsuitable for one reason or another.